La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne virus recognized as a major cause of pediatric encephalitis in North America with 70-130 symptomatic cases each year. The virus was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl who suffered encephalitis and died in La Crosse, Wisconsin. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10-30/100,000 in endemic areas. LAC encephalitis has become the most commonly reported pediatric arboviral encephalitis in the US with 70-130 symptomatic cases a year with severe sequelae. For these reasons we are interested in developing a vaccine to prevent this CNS disease. Sequence analysis: Sequence analysis of the complete LACV genomes of low-passage LACVhuman1960, LACVmosquito1978, and LACVhuman1978 strains and of biologically cloned derivatives of each strain, indicates that circulating LACVs are genetically stable over time and geographic distance with 99.6-100%, 98.9-100%, 97.8-99.6%, and 99.2-99.7% amino acid identity for N, NSs, M polyprotein, and L proteins respectively. We also identified allelic difference in the L, M, and S segments of LACV viruses. Two major alleles (Group 1 and Group 2) were identified with co-segregation of alleles in L, M, and S segments for all but one isolate that was a reassortant virus. This indicated that reassortants occur in nature but they are unusual. Nucleotide sequence analysis of additional human and mosquito isolates revealed that LACV isolated from humans are predominantly from allelic Group 1 that differs from the predominant allele prevalent in mosquitoes, Group 2. Allelic Group 1 viruses are significantly overrepresented in humans (80% of isolates form humans versus only 10% in mosquitoes), and therefore they might be the more virulent of the two groups. An allelic group 1 virus (over-represented in human CNS isolates) was more virulent in the CNS of mice and monkeys than an allelic Group 2 virus. This indicates one of the reasons for the low severe disease to infection ratio for this virus is due to the under-representation of the more virulent virus in nature. Parenthetically, the rhesus monkey model of CNS disease following intracerebral inoculation of LACV can be used to study both the level of attenuation of live attenuated virus vaccine candidates in the CNS and of the efficacy of immunization with such a candidate in a challenge study. A detailed study of the neuropathogenesis of LACV for the CNS of non-human primates is being determined. To identify a nucleotide sequence of LACV associated with the wild type phenotype, i.e. replication competent in insect and mammalian cells and able to cause encephalitic disease in suckling and weanling mice by peripheral and intracranial routes of inoculation, we sequenced the complete genomes of three low-passage LACV isolates, namely, LACV/human/1960, LACV/human/1978, LACV/mosquito/1978, isolated over a 18 year period of time. Biologically cloned derivatives of each virus were also sequenced. The level of neurovirulence and neuroinvasiveness for each of the three virus isolates and their cloned derivatives was determined in mice by assessing clinical disease following intracerebral or intraperitoneal administration of virus. LACV strains appear highly genetically stable in nature, grow to high titers in monkey and mosquito cell cultures, and are highly neurovirulent and neruoinvasive for mice even at low dosage. Since one of the long-term goals of this project is to develop a live attenuated virus vaccine for LACV, the identification of a nucleotide sequence of LACV that specifies a wild type phenotype was seen as an essential first step in this process. A single amino acid substitution was identified in GN (a threonine to alanine substitution at position 148 in the GN) in one of the cloned LACV strains, a strain that exhibited greatly decreased neuroinvasiveness. In addition, a second mutation, a E577G substitution in GC, that attenuates the neuroinvasiveness of LACV for mice has been identified. Such mutations may be useful in developing live-attenuated virus vaccine candidates. These mutations were selected for incorporation into LACV derived using reverse genetics. Reverse genetics: A reverse genetics system for LACV has previously been reported that was used to generate a virus lacking NSs. We have reproduced this system and have generated a set of rLACV viruses with NSs KO, E577G mutation in GC, or T148A mutation in GN either as a single mutation or in various combinations. The rLACV mutants will be evaluated for LD50 and for level of replication in the CNS of mice. Their level of infectivity and immunogenicity will be evaluated in non-human primates to determine if they are infectious, attenuated, and immunogenic for non-human primates. We anticipate that we will be able to attenuate LACV. Given our experience with antigenic chimeric viruses of the various flaviviruses, we sought to explore the possibility that we could use an attenuated LACV as a backbone to create antigenic chimeric viruses in which the coding region of the M segment of LACV is replaced with that of a second bunyavirus. We initiated this study with a bunyavirus from the same California Encephalitis Virus serogroup, but that was serologically sufficiently distant in neutralization assays. The virus selected was Jamestown Canyon Virus (JVC) since it was a BSL2 virus that also can caused disease in humans. JCV is a mosquito-borne virus that has a wide geographic distribution in temperate North America. It causes disease in adult humans ranging from mild febrile disease to severe encephalitis. Its principal host is the white tailed deer and is primarily vectored by Aedes and Ochlerotatus mosquito species. Its seroprevalence is about 4% indicating that infection with this virus is not rare. We have isolated an antigenic chimeric virus and have initiated studies to characterize its properties, comparing its replication in vitro and in vivo with those of its LACV and JCV parents. Preliminary finding indicate that the chimeric virus is attenuated for the CNS of mice.